1. Field of the Invention
The present invention relates to filtration systems. More specifically, the invention relates to a filtration system for fluids, particularly biological fluids. The filtration system includes a filter containing compartment connected at one end to a storage vessel and at the other end to a diaphragm pump. The system creates an alternating tangential flow of fluid through a filter element, a process that offers the benefits of tangential flow without some of its drawbacks. As will become apparent, some of the benefits not offered by other systems include improved processing of fragile materials such as animal cells and biomolecules. Other benefits of the system are embodied in the closed nature of the invention, which among other things allows simplified sanitation and sterilization of the system and allows confinement of biological or some other hazardous material for protection against contamination. Yet other benefits include extended filter life with applications in long term filtration processes such as perfusion of animal cells. Waste fluids may be removed from the culture by filtration, as desired, and fresh fluid may be added to replenish the filtered fluid.
2. Description of the Related Art
Filtration is typically performed to separate, clarify, modify and/or concentrate a fluid solution, mixture or suspension. In the biotechnology and pharmaceutical industries, filtration is vital for the successful production, processing, and testing of new drugs, diagnostics and other biological products. For example, in the process of manufacturing biologicals, using animal cell culture, filtration is done for clarification, selective removal and concentration of certain constituents from the culture media or to modify the media prior to further processing. Filtration may also be used to enhances productivity by maintaining a culture in perfusion at high cell concentration. The invention provides an improved means for fractionating a mixture or suspension of molecules or particulates based on physical and/or chemical properties.
Several specialized filters and filtration methods have been developed to separate materials according to their chemical and physical properties. Filters which have been developed in the art include flat surface filters, pleated filters, multi-unit cassettes, and tubular forms such as hollow fibers. However, many of these filters have short operating lives, and when used to filter cell culture suspension or other biological fluids they tend to clog with dead cells, cell debris, aggregates or other constituents of the fluid. In this regard, U.S. Pat. No. 5,527,467 describes a bioreactor having a one-way rectifying membrane which reduces back filtration of solute molecules.
Sensitivity of many culture media to heat and chemical sterilization precludes the use of some filtration methods. U.S. Pat. No. 4,643,715 describes a medical permeating membrane through which bodily fluids flow in dialysis. U.S. Pat. No. 5,516,431 shows a plasma filtration process for separating blood into blood cells and plasma and the removal of harmful macromolecules. None of the above patents shows filtering with backflushing. U.S. Pat. No. 4,592,848 shows a flow through filter with backflush clearing capability, however, no diaphragm pump is used. U.S. Pat. No. 5,234,605 shows filtering with backflush clearing capability using a diaphragm pump, however, fluids do not traverse back and forth between a fluid storage vessel and a diaphragm pump via an intermediate filter.
Animal cells grow substantially slower than most microorganisms, and lacking protective cell wall, they are also more fragile. Some known methods for increasing the productivity of microbial culture production including increasing agitation rates and vigorous delivery of gases into the culture are not feasible with animal cells. Thus, production is limited to very gentle culture conditions and low cell concentrations. One way to increase the cell concentration, yet maintain gentle culture conditions is through the perfusion method.
In the perfusion method for growing cells, culture medium, whose nutrients have been consumed and which contains increased levels of harmful waste products, is continuously removed from the culture and replaced with fresh medium. The constant addition of fresh medium while eliminating waste products provides the cells with the nutrients it requires to achieve high cell concentrations. Unlike the constant changing conditions during batch culture method of production, the perfusion method offers the means to achieve and maintain a culture in steady state.
In normal batch cultures production processes, cells are first inoculated into a fresh medium and the cells rapidly enter a log grow phase. As they consume the medium nutrients and waste products accumulate, the cells transition to a stationary followed by a decay phase. While several methods have been developed to optimize batch culture production, in each case, these processes undergo rapid growth and decay cycles. In perfusion, however, since waste products generated by the culture are continuously removed and the culture is continuously replenished with fresh medium, it is possible to achieve a state of equilibrium in which cell concentration and productivity are maintained. Typically, about one culture volume is exchanged per day and the cell concentration achieved in perfusion are typically 2 to more than 10 times that achieved at the peak of batch culture.
Despite the potential benefits of the perfusion method, it has gained only modest acceptance. One key reason is due to the low reliability of currently available perfusion devices. Presently known perfusion methods which are used to separate a medium from cells frequently damage the cells. This damage may result from direct physical disruption by shearing forces of the system, depletion of nutrients in the medium, changes in physiological conditions of the culture, such as ionic strength, pH, etc., exposure to growth suppressing elements released by the cells. The resulting build up of dead cells and aggregates on screens or filters, resulting in clogging and failure of the perfusion device. At high cell concentrations, typical of perfusion cultures, these problems may be amplified. This is particularly the case with a number of perfusion devices which are contained inside the process vessel and can not be replaced during a production run. Should such an internal system fail, the entire production run must terminated.
The xe2x80x9cspin basketxe2x80x9d system is one type of internal perfusion device. This method uses a basket, which may contain an agitation impeller on the bottom center axis. The perimeter surface of the basket is covered by a mesh screen, with about 20 micron pore opening. Rotation of the basket inhibits the attachment of cells to the screen or penetration through the screen into the basket. Waste medium removed from within the basket is replaced by addition of fresh medium to the culture. This system is limited, however, because cells and cell debris gradually do accumulate on the screen, reducing the screen""s ability to fractionate the cells from the medium. Eventually insufficient medium can be removed from the system to maintain an adequate perfusion rate. The culture deteriorate as it becomes increasingly deprived of nutrients.
The use of flat filters and xe2x80x9cplate and framexe2x80x9d systems have limited usefulness in perfusion applications since such systems are difficult to sterilize or keep sterile. Furthermore, maintaining uniform flow across the entire rectangular cross section of the filter is somewhat difficult. Other perfusion devices based on cell settling have not been used extensively because of limited scale up potential and the nonhomogeneous nature of the settling device. Cells confined to the settling device may be deprived of essential nutrients, primarily oxygen.
In one type of external filtration perfusion systems, a culture medium is circulated from a vessel, through a hollow fiber cartridge and back to the vessel. A pump attached to the tubing between hollow fiber and vessel circulates the culture content from the vessel, through the hollow fiber cartridge and back to the vessel. The process produces tangential flow across the hollow fiber membranes. A second pump on the filtrate side of the hollow fiber cartridge controls the rate of filtrate removal. The use of hollow fiber is preferred over flat sheet, plate and frame, type systems because, unlike the later, the enclosed nature of the hollow fiber module is simpler to sterilize and maintain sterile, uniform flow can be generated across the entire cross section of the hollow fiber module. One may also achieve uniform scale-up by a proportional increase in the number of hollow fibers. However, like the spin-basket method, the hollow fiber filters are prone to clogging by accumulation of particulates and gelatin on the membrane surface. Recirculation in one direction through the hollow fiber cartridge typically results in clogging of the hollow fiber lumen by aggregates lodging at lumen inlet. Such aggregates may grow in size and as more hollow fibers are blocked, filtration capacity declines.
It would therefore be desirable to create a filtration system where waste medium or fluid is continuously removed and the fluid is continuously replenished with fresh medium. It would also be desirable to create a filtration system which creates an alternating tangential flow which continuously filters fluids, such as biological fluids with minimal damage to cells or other constituents of a particular process, which minimizes clogging, that may be replaced in mid process with minimum disruption of the process, that may be sterilized in all parts and maintain sterile, that may contain only a single connection to the process vessel and that may be adaptable to most process.
The present invention provides a solution to these problems. It includes a filter containing compartment connected at one end to a storage vessel and at the other end to a diaphragm pump. The pump circulates a fluid from the vessel through the filter element and to the pump. The flow is then reversed, and the fluid is circulated back from the pump through the filter element and to the vessel. Thus, an alternating tangential flow of fluid is produced across the filter element. Furthermore, uniform flow can be generated across the entire filter. Thus, this system thus provides a means for generating rapid, low shear, tangential flow. The process is also advantageous for maintaining since the system can be sterilized without terminating a production run. Hollow fiber (HF) type filters afford longer operating lives, and they are available in many sizes, configurations, materials, pore sizes and porosity. Furthermore, the process need not be limited to the use of hollow fiber filters. It is possible to insert other separation devices in the hollow fiber housing. One such device is screen module, consisting of a screen mash as the separation matrix. All such separation modules will be referred to, collectively, as the filter element or simply as the filter. Additional advantages not offered by other filtration systems will become apparent to those skilled in the art upon a consideration of the configuration to be described.
The invention provides a fluid filtration system comprising:
a) at least one fluid storage vessel;
b) at least one filter containing compartment;
c) a fluid connector attached to the storage vessel and to an entrance end of the filter containing compartment, which connector is capable of directing a fluid from the storage vessel into the entrance end of the filter containing compartment;
d) at least one diaphragm pump connected at an exit end of the filter containing compartment; which diaphragm pump is capable of alternately receiving fluid from the exit end of the filter containing compartment and then expelling the fluid back into the exit end of the filter containing compartment; and
e) at least one fluid harvest port connected to the filter containing compartment for removing filtered fluid from the filter containing compartment.
The invention further provides a process for filtering a fluid comprising:
a) providing a fluid filtration system comprising at least one fluid storage vessel;
at least one filter containing compartment; a fluid connector attached to the storage vessel and to an entrance end of the filter containing compartment, which connector is capable of directing a fluid from the storage vessel into the entrance end of the filter containing compartment;
at least one diaphragm pump connected at an exit end of the filter containing compartment; which diaphragm pump is capable of alternately receiving fluid from the exit end of the filter containing compartment and then expelling the fluid back into the exit end of the filter containing compartment; and at least one fluid harvest port connected to the filter containing compartment for removing filtered fluid from the filter containing compartment;
b) filtering a fluid by causing the fluid to flow from the storage vessel through the filter containing compartment and then to the diaphragm pump;
c) re-filtering the fluid by causing at least a portion of the fluid to flow from the diaphragm pump through the filter containing compartment and then to the storage vessel;
d) optionally repeating steps b and c; and
e) removing the filtered fluid from the filtration system.
The invention also provides a process for sterilizing a fluid filtration system comprising:
a) providing a fluid filtration system comprising at least one fluid storage vessel;
at least one filter containing compartment; a fluid connector attached to the storage vessel and to an entrance end of the filter containing compartment, which connector is capable of directing a fluid from the storage vessel into the entrance end of the filter containing compartment;
at least one diaphragm pump connected at an exit end of the filter containing compartment; which diaphragm pump is capable of alternately receiving fluid from the exit end of the filter containing compartment and then expelling the fluid back into the exit end of the filter containing compartment; and at least one fluid harvest port connected to the filter containing compartment for removing filtered fluid from the filter containing compartment;
b) injecting steam into at least a portion of the fluid filtration system via at least one steam inlet; and
c) removing the steam from the fluid filtration system via at least one steam outlet.
The invention still further provides a fluid filtration system comprising:
a) at least one fluid storage vessel;
b) at least one filter containing compartment;
c) a fluid connector attached at one end thereof to the storage vessel by a valve, and attached at another end thereof to an entrance end of the filter containing compartment by a valve, which connector is capable of directing a fluid from the storage vessel into the entrance end of the filter containing compartment; said fluid connector having a steam injection port and a condensate outlet;
d) at least one diaphragm pump connected at an exit end of the filter containing compartment; which diaphragm pump is capable of alternately receiving fluid from the exit end of the filter containing compartment and expelling the fluid back into the exit end of the filter containing compartment; said diaphragm pump having a pump housing comprising a first and a second chamber separated by a diaphragm; the first chamber of the diaphragm pump being connected to a gas port capable of alternately injecting a gas into and out of the first chamber; the second chamber being in fluid flow cooperation with the exit end of the filter containing compartment; a controller for controlling the movement of the diaphragm within the pump housing; a fluid sampling port attached through a wall of the second chamber;
e) at least one fluid harvest port connected to the filter containing compartment for removing filtered fluid from the filter containing compartment, said harvest port being connected via a fluid flow line to a fluid pump; first and second fluid control valves attached in series between the harvest port and the fluid pump; a steam injection port and a condensate outlet attached to the fluid flow line between the steam injection port and a condensate outlet;
f) a pressure dampener attached through a wall of the filter containing compartment;
g) wherein the filter which comprises a plurality of bundled hollow fibers whose axes extend longitudinally from the entrance end to the exit end of the filter containing compartment.